FIG. 4.

Effects of aging on generation of the sulfinic forms of Prx enzymes or GAPDH. (A) Fresh RBCs were separated into four fractions (F1, F2, F3, and F4) on the basis of their density (age), and lysates of each fraction were subjected to immunoblot analysis with antibodies specific for the sulfinic form of 2-Cys Prxs, Prx VI, or GAPDH. Equal loading of proteins was confirmed by immunoblot analysis with antibodies to Prx II, to Prx VI, and to GAPDH, respectively. Immunoblot analysis was also performed with antibodies to Srx. The band intensities are average of two independent experiments. (B) Lysates of the age-related fractions of RBCs were also subjected to two-dimensional PAGE on a 13-cm Immobiline DryStrips (Amersham; pH 4 to 7, linear) and probed by immunoblot analysis with antibodies to Prx II(20). The positions of hyperoxidized (Ox) and reduced (Re) forms of Prx II are indicated.